Transglutaminases (TGases) catalyze the formation of a cross-link between a donor amide group of a protein-bound glutamine residue and an acceptor epsilon-NH2 of a protein-bound lysine residue. This cross-link is an isopeptide bond that cannot be cleaved in vertebrate organisms. The net result therefore is the formation of a permanent, stable, insoluble macromolecular protein complex. In the epidermis and other stratified squamous epithelia, several of the nine known TGase enzymes are expressed. In particular, TGases 1, 2 and 3 cross-link a variety of defined structural proteins to form the cornified cell envelope which is a principal component of epithelial barrier function. We are studying in detail each of these enzymes, and their roles in diseases. Transglutaminase 1 The TGase 1 enzyme in cultured keratinocytes or foreskin epidermal cells is complex since it exists in multiple soluble and membrane-bound full-length as well as proteolytically-processed forms. Most of the enzyme is membrane bound by way of myristate and anchorages on the amino-terminal segment which is unique to the TGase 1 enzyme. The various forms display wide variations in specific activities, but these are difficult to measure because the enzyme is inherently unstable and easily degraded by proteolysis. To address structural and functional questions, we have been successful in expression in baculovirus systems. Previous work from this laboratory has shown that mutations in the TGM1 gene, encoding the TGase 1 enzyme, cause the autosomal recessive disorder lamellar ichthyosis. We examined its molecular basis in a Japanese family and reported two novel TGM1 mutations (R348X, and Y365D). Molecular docking studies revealed that the LI phenotype of this proband can be explained by the prediction that each of the maternally and paternally derived mutations will result in loss of enzyme activity. This study was published. In another investigation of this kind, two self-healing collodion baby siblings with markedly diminished epidermal TGM1 activity we were found to have compound heterozygous TGM1 mutations G278R and D490G. Molecular modeling and biochemical assays of mutant proteins under elevated hydrostatic pressure suggest significantly reduced activity in G278R and a chelation of water molecules in D490G that locks the mutated enzyme in an inactive trans conformation in utero. After birth these water molecules are removed and the enzyme is predicted to isomerize back to a partially active cis form, explaining the dramatic improvement of this skin condition. This study was also published With the demise of the former Principal Investigator, Dr Peter Steinert, in spring 2003, activity on this project in the LSB wound down in FY04 and is essentially erminated. The crystallographic studies of several transglutaminases that were initiated in the LSB are being continued by Dr Bijan Ahvazi, now in the X-ray crystallography unit of NIAMS, as reported in AR04-114201. Several papers on these structures and on modelling based on these structures were published during FY04, as were several papers reporting genetic studies relating to phenotypes of mutations in transglutaminase genes.